In the field of molecular diagnostics, the amplification of nucleic acids from numerous sources has been of considerable significance. Examples for diagnostic applications of nucleic acid amplification and detection are the detection of viruses such as Human Papilloma Virus (HPV), West Nile Virus (WNV) or the routine screening of blood donations for the presence of Human Immunodeficiency Virus (HIV), Hepatitis-B (HBV) and/or C Virus (HCV). Furthermore, said amplification techniques are suitable for bacterial targets such as mycobacteria or Chlamydia trachomatis and Neisseria gonorrhoeae, or the analysis of oncology markers.
The most prominent and widely-used amplification technique is Polymerase Chain Reaction (PCR). Other amplification reactions comprise, among others, the Ligase Chain Reaction, Polymerase Ligase. Chain Reaction, Gap-LCR, Repair Chain Reaction, 3SR, NASBA, Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), and Qβ-amplification.
Automated systems for PCR-based analysis often make use of real-time detection of product amplification during the PCR process in the same reaction vessel. Key to such methods is the use of modified oligonucleotides carrying reporter groups or labels.
It has been shown that amplification and detection of more than one target nucleic acid in the same vessel is possible. This method is commonly termed “multiplex” amplification and requires different labels for distinction if real-time detection is performed.
Rohayem et al. have suggested a multiplex RT-PCR assay for the agarose-gel-based detection of the RNA viruses Norovirus and Astrovirus and the DNA virus Adenovirus (2004, Journal of Virological Methods 118, 49-59).
An improved method for simultaneous amplification and detection of different nucleic acids is provided.